Crystalline freebase forms of a biphenyl compound

ABSTRACT

The invention provides two crystalline freebase forms of biphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester. The invention also provides pharmaceutical compositions comprising the crystalline freebase or prepared using the crystalline freebases; processes and intermediates for preparing the crystalline freebases; and methods of using the crystalline freebases to treat a pulmonary disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/677,264, filed on Aug. 15, 2017; which application is a continuationof U.S. application Ser. No. 15/206,877, filed on Jul. 11, 2016 (nowU.S. Pat. No. 9,765,028); which application is a continuation of U.S.application Ser. No. 14/955,515, filed on Dec. 1, 2015 (now U.S. Pat.No. 9,415,041); which application is a continuation of U.S. applicationSer. No. 14/547,455, filed on Nov. 19, 2014 (now U.S. Pat. No.9,226,896); which application is a continuation of U.S. application Ser.No. 13/973,174, filed Aug. 22, 2013 (now U.S. Pat. No. 8,921,396); whichapplication is a divisional of U.S. application Ser. No. 12/835,964,filed Jul. 14, 2010 (now U.S. Pat. No. 8,541,451); which applicationclaims the benefit of U.S. Provisional Application No. 61/225,803, filedon Jul. 15, 2009; the entire disclosures of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel crystalline forms of a biphenylcompound, which are expected to be useful for treating pulmonarydisorders. The invention also relates to pharmaceutical compositionscomprising the crystalline compounds or prepared from such compounds,processes and intermediates for preparing such crystalline compounds andmethods of using such compounds to treat a pulmonary disorder.

State of the Art

U.S. Patent Publication No. 2005/0203133 to Mammen et al. disclosesnovel biphenyl compounds that are expected to be useful for treatingpulmonary disorders such as chronic obstructive pulmonary disease (COPD)and asthma. In particular, the compound biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester is specifically described in this application as possessingmuscarinic receptor antagonist or anticholinergic activity.

The chemical structure of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester isrepresented by formula I:

The compound of formula I has been named using thecommercially-available AutoNom software (MDL, San Leandro, Calif.).

Therapeutic agents useful for treating pulmonary or respiratorydisorders are advantageously administered directly into the respiratorytract by inhalation. In this regard, several types of pharmaceuticalinhalation devices have been developed for administering therapeuticagents by inhalation including dry powder inhalers (DPI), metered-doseinhalers (MDI) and nebulizer inhalers. When preparing pharmaceuticalcompositions and formulations for use in such devices, it is highlydesirable to have a crystalline form of the therapeutic agent that isneither hygroscopic nor deliquescent and which has a relatively highmelting point thereby allowing the material to be micronized withoutsignificant decomposition. Although crystalline freebase forms of thecompound of formula I have been reported in U.S. Patent Publication No.2007/0112027 to Axt et al. as Form I and Form II, the crystallinefreebase forms of the present invention have different and particularlyuseful properties, including higher melting points.

SUMMARY OF THE INVENTION

One aspect of the invention relates to crystalline freebase forms ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester characterized by a powder x-ray diffraction patterncomprising diffraction peaks at 2θ values of 6.6±0.1, 13.1±0.1,18.6±0.1, 19.7±0.1, and 20.2±0.1.

Another aspect of the invention relates to a crystalline freebase ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester, designated as form III, which is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 6.6±0.1, 13.1±0.1, 18.6±0.1, 19.7±0.1, and 20.2±0.1; andfurther characterized by having five or more additional diffractionpeaks at 2θ values selected from 8.8±0.1, 10.1±0.1, 11.4±0.1, 11.6±0.1,14.8±0.1, 15.2±0.1, 16.1±0.1, 16.4±0.1, 16.9±0.1, 17.5±0.1, 18.2±0.1,19.3±0.1, 19.9±0.1, 20.8±0.1, 21.1±0.1, 21.7±0.1, and 22.3±0.1.

Still another aspect of the invention relates to a crystalline freebaseof biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester, designated as form IV, which is characterized by apowder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 6.6±0.1, 13.1±0.1, 18.6±0.1, 19.7±0.1, and 20.2±0.1; andfurther characterized by having five or more additional diffractionpeaks at 2θ values selected from 10.6±0.1, 15.0±0.1, 16.0±0.1, 17.3±0.1,17.7±0.1, 20.9±0.1, 21.4±0.1, 22.6±0.1, 24.6±0.1, and 27.8±0.1.

Another aspect of the invention relates to pharmaceutical compositioncomprising a crystalline freebase of the invention and apharmaceutically acceptable carrier. Yet another aspect of the inventionrelates to pharmaceutical compositions comprising a crystalline freebaseof the invention in combination with one or more other therapeuticagents. Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising (a) a pharmaceutically acceptablecarrier and a therapeutically effective amount of a crystalline freebaseof the invention; and (b) a therapeutically effective amount of an agentselected from a steroidal anti-inflammatory agent such as acorticosteroid; a β₂ adrenergic receptor agonist; a phosphodiesterase-4inhibitor; or a combination thereof; wherein the crystalline freebaseand the agent are formulated together or separately. When the agent isformulated separately, a pharmaceutically acceptable carrier may beincluded. Typically, the crystalline freebase of the invention and theagent will be present in therapeutically effective amounts.

Another aspect of the invention relates to a pharmaceutical compositioncomprising an aqueous isotonic saline solution comprising a crystallinefreebase of the invention, wherein the solution has a pH in the range offrom about 4 to 6. In a particular embodiment, an aqueous nebulizerformulation is buffered with citrate buffer to a pH of about 5.

In one embodiment, this invention relates to a drug delivery devicecomprising a dry powder inhaler containing a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a crystallinefreebase of the invention.

The compound of formula I has muscarinic receptor antagonist activity.Accordingly, a crystalline freebase of the compound of formula I isexpected to have the same activity, and thus find utility in treatingpulmonary disorders such as asthma and chronic obstructive pulmonarydisease. Thus, another aspect of the invention relates to a method fortreating a pulmonary disorder comprising administering to a patient atherapeutically effective amount of a crystalline freebase of theinvention. Still another aspect of the invention relates to a method ofproducing bronchodilation in a patient comprising administering to thepatient a bronchodilation-producing amount of a crystalline freebase ofthe invention. In one embodiment, the compound is administered byinhalation. The invention also provides a method of treating chronicobstructive pulmonary disease or asthma comprising administering to apatient a therapeutically effective amount of a crystalline freebase ofthe invention. Another aspect of the invention relates to a method forantagonizing a muscarinic receptor in a mammal comprising administeringto the mammal a therapeutically effective amount of a crystallinefreebase of the invention.

The invention also relates to processes for preparing crystallinefreebase forms of the compound of formula I. The invention also providesa process for purifying the compound of formula I comprising forming acrystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester. The invention further relates to products preparedby the processes described herein.

The invention also relates to a crystalline freebase of the compound offormula I in a micronized form; and to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a micronizedcrystalline freebase of the invention.

The invention also relates to crystalline freebase forms of the compoundof formula I for use in therapy or as a medicament. Additionally, theinvention relates to use of a crystalline freebase of the invention forthe manufacture of a medicament; especially for the manufacture of amedicament for the treatment of a pulmonary disorder or for antagonizinga muscarinic receptor in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of Form III ofthe crystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (the compound of formula I). Other characteristics of Form III arepresented in FIG. 4, which shows a differential scanning calorimetry(DSC) thermogram and FIG. 6, which shows a thermal gravimetric analysis(TGA) trace.

FIG. 2 shows a PXRD pattern of Form IV of the crystalline freebase ofthe compound of formula I. FIG. 3 shows an overlay of the PXRD patternsof Form III and Form IV. Other characteristics of Form IV are presentedin FIG. 5, which shows a DSC thermogram, and FIG. 7, which shows a TGAtrace.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides crystalline freebase forms ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (formula I). Surprisingly, the crystalline freebase forms of theinvention have been found not to be deliquescent, even when exposed toatmospheric moisture. Additionally, the crystalline freebase forms ofthe invention have acceptable levels of hygroscopicity and acceptablemelting points. For example, the crystalline freebase Form III has amelting point of about 125° C. and the crystalline freebase Form IV hasa melting point of about 119° C.

Among other uses, the crystalline freebase forms of the invention areuseful for preparing pharmaceutical compositions expected to haveutility in treating pulmonary disorders. Accordingly, one aspect of theinvention relates to a pharmaceutical composition comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of a crystalline freebase of the invention.

Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

Both Form III and Form IV are anhydrous freebase crystal polymorphs.When reference is made to “a crystalline freebase of the invention”, itis understood that the term includes Form III and Form IV.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug Administration.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof,i.e., the amount of drug needed to obtain the desired therapeuticeffect. For example, a therapeutically effective amount for treating apulmonary disorder is an amount of compound needed to, for example,reduce, suppress, eliminate or prevent the symptoms of asthma or chronicobstructive pulmonary disease (“COPD”), or to treat the underlying causeof asthma or COPD. In one embodiment, a therapeutically effective amountis that amount needed to produce bronchodilation. On the other hand, theterm “effective amount” means an amount sufficient to obtain a desiredresult, which may not necessarily be a therapeutic result. For example,when studying a system comprising a muscarinic receptor, an “effectiveamount” may be the amount needed to antagonize the receptor.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patientsuch as a mammal (particularly a human) that includes: (a) preventingthe disease or medical condition from occurring, that is, prophylactictreatment of a patient; (b) ameliorating the disease or medicalcondition such as by eliminating or causing regression of the disease ormedical condition in a patient; (c) suppressing the disease or medicalcondition such as by slowing or arresting the development of the diseaseor medical condition in a patient; or (d) alleviating the symptoms ofthe disease or medical condition in a patient. For example, the term“treating COPD” would include preventing COPD from occurring,ameliorating COPD, suppressing COPD, and alleviating the symptoms ofCOPD. The term “patient” is intended to include those mammals, such ashumans, that are in need of treatment or disease prevention or that arepresently being treated for disease prevention or treatment of aspecific disease or medical condition. The term “patient” also includestest subjects in which compounds of the invention are being evaluated ortest subjects being used in a assay, for example an animal model.

Synthesis

The crystalline freebase forms of the invention can be synthesized fromreadily available starting materials as described below and in theExamples. While there may be several methods that can be used to produceeach crystalline freebase form, it is noted, however, that thecrystalline content as well as the habit of the crystals (size andshape) may vary, based partly upon the method of preparation, as well ason the solvent composition.

It will be appreciated that while specific process conditions (i.e.crystallization temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. In some instances, reactions orcrystallizations were conducted at room temperature and no actualtemperature measurement was taken. It is understood that roomtemperature can be taken to mean a temperature within the range commonlyassociated with the ambient temperature in a laboratory environment, andwill typically be in the range of about 25° C. to about 50° C. In otherinstances, reactions or crystallizations were conducted at roomtemperature and the temperature was actually measured and recorded. Allweights, volumes and equivalents are relative to thebiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (or salt form) starting material.

Generally, the crystallizations are conducted in a suitable inertdiluent or solvent system, examples of which include, but are notlimited to, methanol, ethanol, isopropanol, isobutanol, ethyl acetate,acetonitrile, dichloromethane, methyl t-butyl ether, and the like, andmixtures thereof. Upon completion of any of the foregoingcrystallizations, the crystalline compounds can be isolated from thereaction mixture by any conventional means such as precipitation,concentration, centrifugation and the like.

The biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester, as well as its salts such as the diphosphate salt, employed inthe invention can be readily prepared from commercially availablestarting materials and reagents using the procedures described in theExamples, or using the procedures described in U.S. Patent PublicationNo. 2005/0203133 to Mammen et al. and U.S. Patent Publication No.2007/0112027 to Axt et al.

The molar ratios described in the methods of the invention can bereadily determined by various methods available to those skilled in theart. For example, such molar ratios can be readily determined by ¹H NMR.Alternatively, elemental analysis and HPLC methods can be used todetermine the molar ratio.

Form III

Form III crystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoyl-piperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester can be prepared from the ester or the diphosphate salt of theester.

In one embodiment, the Form III crystalline freebase is prepared bycontacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]-methylamino}ethyl)piperidin-4-ylester with acetonitrile. Typically, the ratio of milligrams of the esterto total milliliters of acetonitrile is about 100:1, with theacetonitrile being added in two steps. Generally, this reaction isconducted while repeatedly cycling through a temperature range of 0-40°C. The solids are then isolated by vacuum filtration and dried.

In another embodiment, the Form III crystalline freebase is preparedusing a seed crystal of the Form III crystalline freebase and thediphosphate salt of the ester. This method involves: a) forming a seedcrystal of the crystalline freebase Form III; b) dissolving thediphosphate salt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester in isopropyl acetate and water to form a solution; c) and addingthe seed crystal to the solution. More specifically, the diphosphatesalt of the ester (1 wt) is slurried in isopropyl acetate (17.5 vol) andwater (10 vol) at 20±3° C. under nitrogen. The suspension is warmed to53±3° C. and 10M NaOH (0.5 vol) is added. The mixture is stirred at thattemperature for a short time, then the layers are separated and thebasic aqueous layer is removed. Water (5 vol) is added to the organiclayer, and stirred. The layers are separated and the water layer isremoved. Isopropyl acetate (17.5 vol) is added and about 10 volumes ofdistillate are collected by atmospheric distillation. This step isrepeated with additional isopropyl acetate (10 vol). After the seconddistillation, the temperature of the clear solution is reduced to 53±3°C., then seeded with a suspension of crystalline freebase Form III(0.005 wt; 0.5 wt %) in isopropyl acetate (0.08 vol). The resultingsuspension is stirred at 53±3° C. for at least 2 hours, then cooled to10±3° C. at an approximate cooling rate of 0.19° C./min. The suspensionis stirred at 10±3° C. for at least 2 hours and then is collected byfiltration. The resulting filter cake is washed with isopropyl acetate(2×3 volumes) and the product is then dried to yield the Form IIIcrystalline freebase.

Form IV

In one embodiment, the Form IV crystalline freebase is prepared using aseed crystal of the Form III crystalline freebase. This method involves:a) forming a seed crystal of the crystalline freebase Form III; b)dissolving the crystalline freebase Form III in acetonitrile to form asolution; c) and adding the seed crystal to the solution. Typically, theweight ratio of seed to ester is in the range of about 2:250. Typically,the ratio of grams of crystalline freebase Form III to total millilitersof acetonitrile is within the range of about 2:10 to 3:30, with 2.5:16being one range. The acetonitrile is usually added in several aliquots.Generally, this reaction is conducted while repeatedly cycling through atemperature range of 0-40° C. The solids are then isolated by vacuumfiltration and dried.

Crystalline Properties

As is well known in the field of powder x-ray diffraction, relative peakheights of powder x-ray diffraction (PXRD) spectra are dependent on anumber of factors relating to sample preparation and instrumentgeometry, while peak positions are relatively insensitive toexperimental details. PXRD patterns for the crystalline freebase FormIII and Form IV were obtained as set forth in Example 5. Thus, in oneembodiment, a crystalline compound of the invention is characterized bya PXRD pattern having certain peak positions.

Each crystalline freebase form of the invention exhibits a differentPXRD pattern, but with certain common peaks. Thus, in one embodiment,the invention relates to crystalline freebase forms ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester characterized by a powder x-ray diffraction patterncomprising diffraction peaks at 2θ values selected from 6.6±0.1,13.1±0.1, 18.6±0.1, 19.7±0.1, and 20.2±0.1.

In one embodiment, the crystalline freebase Form III is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 6.6±0.1, 13.1±0.1, 18.6±0.1, 19.7±0.1, and 20.2±0.1; andfurther characterized by having five or more additional diffractionpeaks at 2θ values selected from 8.8±0.1, 10.1±0.1, 11.4±0.1, 11.6±0.1,14.8±0.1, 15.2±0.1, 16.1±0.1, 16.4±0.1, 16.9±0.1, 17.5±0.1, 18.2±0.1,19.3±0.1, 19.9±0.1, 20.8±0.1, 21.1±0.1, 21.7±0.1, and 22.3±0.1. Inanother embodiment, the crystalline freebase Form III is characterizedby a powder x-ray diffraction comprising diffraction peaks at 2θ valuesselected from 6.6±0.1, 11.4±0.1, 13.1±0.1, 16.1±0.1, 17.5±0.1, 18.2±0.1,18.6±0.1, 19.3±0.1, 19.7±0.1, 19.9±0.1, 20.2±0.1, 20.8±0.1, 21.1±0.1,21.7±0.1, and 22.3±0.1. In yet another embodiment, the crystallinefreebase Form III is characterized by a PXRD pattern in which the peakpositions are substantially in accordance with those shown in FIG. 1.

In one embodiment, the crystalline freebase Form IV is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 6.6±0.1, 13.1±0.1, 18.6±0.1, 19.7±0.1, and 20.2±0.1; andfurther characterized by having five or more additional diffractionpeaks at 2θ values selected from 10.6±0.1, 15.0±0.1, 16.0±0.1, 17.3±0.1,17.7±0.1, 20.9±0.1, 21.4±0.1, 22.6±0.1, 24.6±0.1, and 27.8±0.1. Inanother embodiment, the crystalline freebase Form IV is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues selected from 6.6±0.1, 13.1±0.1, 15.0±0.1, 17.3±0.1, 17.7±0.1,18.6±0.1, 19.7±0.1, 20.2±0.1, 20.9±0.1, 21.4±0.1, and 22.6±0.1. In yetanother embodiment, the crystalline freebase Form IV is characterized bya PXRD pattern in which the peak positions are substantially inaccordance with those shown in FIG. 2.

In yet another embodiment, a crystalline freebase of the invention ischaracterized by a differential scanning calorimetry (DSC) thermogram.DSC thermograms were obtained as set forth in Example 6. Melting pointsreported herein are estimated on the basis of the melt onset registeredduring DSC analysis. Thus, in one embodiment, a crystalline compound ofthe invention is characterized by its DSC thermograph. In oneembodiment, the crystalline freebase Form III is characterized by a DSCthermograph which shows an onset of endothermic heat flow at about 123°C. and a melting point of about 125° C., as seen in FIG. 4. In anotherembodiment, the crystalline freebase Form IV is characterized by a DSCthermograph which shows one onset of endothermic heat flow at about 66°C., a second onset of endothermic heat flow at about 119° C., and amelting point of about 119° C., as seen in FIG. 5.

Thermogravimetric analysis (TGA) was performed on the crystallinefreebase forms of the invention as described in Example 6. Thus, in oneembodiment, a crystalline freebase is characterized by its TGA trace. Inone embodiment, the crystalline freebase Form III is characterized bythe TGA trace depicted in FIG. 6. In another embodiment, the crystallinefreebase Form IV is characterized by the TGA trace depicted in FIG. 7.

A gravimetric vapor sorption (GVS) assessment was performed on thecrystalline freebase forms of the invention as described in Example 7.The crystalline freebase forms of the invention have been demonstratedto have a reversible sorption/desorption profiles with acceptable levelsof hygroscopicity. For example, the crystalline freebase Form III showeda reversible water uptake of <2% wt/wt between 0 and 90% relativehumidity at 25° C. Additionally, the crystalline freebase Form III hasbeen found to be stable upon exposure to elevated temperature andhumidity.

These properties of the crystalline freebase forms of the invention arefurther illustrated in the Examples below.

Utility

The compound of formula I possesses muscarinic receptor antagonistactivity and therefore, a crystalline freebase form of the compound offormula I is expected to be useful for treating medical conditionsmediated by muscarinic receptors, i.e., medical conditions that areameliorated by treatment with a muscarinic receptor antagonist. Suchmedical conditions include, by way of example, pulmonary disorders ordiseases including those associated with reversible airway obstructionsuch as chronic obstructive pulmonary disease (e.g., chronic and wheezybronchitis and emphysema), asthma, pulmonary fibrosis, allergicrhinitis, rhinorrhea, and the like. Other medical conditions that can betreated with muscarinic receptor antagonists are genitourinary tractdisorders such as overactive bladder or detrusor hyperactivity and theirsymptoms; gastrointestinal tract disorders such as irritable bowelsyndrome, diverticular disease, achalasia, gastrointestinalhypermotility disorders and diarrhea; cardiac arrhythmias such as sinusbradycardia; Parkinson's disease; cognitive disorders such asAlzheimer's disease; dismenorrhea; and the like.

Accordingly, in one embodiment, the invention relates to a method fortreating a pulmonary disorder, the method comprising administering to apatient a therapeutically effective amount of a crystalline freebase ofbiphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester.When used to treat a pulmonary disorder, a crystalline freebase of theinvention will typically be administered by inhalation in multiple dosesper day, in a single daily dose or a single weekly dose. Generally, thedose for treating a pulmonary disorder will range from about 10 μg/dayto 200 μg/day.

When administered by inhalation, a crystalline freebase of the inventiontypically will have the effect of producing bronchodilation.Accordingly, in another embodiment, the invention relates to a method ofproducing bronchodilation in a patient, the method comprisingadministering to a patient a bronchodilation-producing amount of acrystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester. Generally, the therapeutically effective dose for producingbronchodilation will range from about 10 μg/day to 200 μg/day.

In one embodiment, the invention relates to a method of treating chronicobstructive pulmonary disease or asthma, the method comprisingadministering to a patient a therapeutically effective amount of acrystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester. When used to treat a COPD or asthma, a crystalline freebase ofthe invention will typically be administered by inhalation in multipledoses per day or in a single daily dose. Generally, the dose fortreating COPD or asthma will range from about 10 μg/day to 200 μg/day.As used herein, COPD includes chronic obstructive bronchitis andemphysema (see, for example, Barnes, Chronic Obstructive PulmonaryDisease, New England Journal of Medicine 343:269-78 (2000)).

When used to treat a pulmonary disorder, a crystalline freebase of theinvention is optionally administered in combination with othertherapeutic agents. Accordingly, in a particular embodiment, thepharmaceutical compositions and methods of the invention furthercomprise a therapeutically effective amount of a β₂-adrenoreceptoragonist, a corticosteroid, a non-steroidal anti-inflammatory agent, orcombination thereof.

In another embodiment, a crystalline freebase of the invention is usedto antagonize a muscarinic receptor in biological system, and a mammalin particular such as mice, rats, guinea pigs, rabbits, dogs, pigs,humans and so forth. In this embodiment, a therapeutically effectiveamount of a crystalline freebase of the invention is administered to themammal. If desired, the effects of antagonizing the muscarinic receptorcan then determined using conventional procedures and equipment.

The properties and utility of a crystalline freebase of the invention,such as the muscarinic receptor antagonizing activity, can bedemonstrated using various in vitro and in vivo assays that arewell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples andinclude by way of illustration and not limitation, assays that measurehM₁, hM₂, hM₃, hM₄, and hM₅ muscarinic receptor binding (for example, asdescribed in Assay 1). Useful functional assays to determine themuscarinic receptor antagonizing activity of a crystalline freebase ofthe invention include by way of illustration and not limitation, assaysthat measure ligand-mediated changes in intracellular cyclic adenosinemonophosphate (cAMP), ligand-mediated changes in activity of the enzymeadenylyl cyclase (which synthesizes cAMP), ligand-mediated changes inincorporation of guanosine 5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) intoisolated membranes via receptor catalyzed exchange of [³⁵S]GTPγS forGDP, ligand-mediated changes in free intracellular calcium ions(measured, for example, with a fluorescence-linked imaging plate readeror FLIPR® from Molecular Devices, Inc.), and the like. Exemplary assaysare described in Assay 2. The crystalline freebase is expected toantagonize or decrease the activation of muscarinic receptors in any ofthe assays listed above, or assays of a similar nature, and willtypically be used in these studies at a concentration ranging from about0.1-100 nanomolar. Thus, the aforementioned assays are useful indetermining the therapeutic utility, for example, the bronchodilatingactivity, of a crystalline freebase of the invention.

Other properties and utilities of a crystalline freebase of theinvention can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, the in vivo potencyof a crystalline freebase can be measured in an animal model such as theEinthoven model. Briefly, the bronchodilator activity of a crystallinefreebase is evaluated in an anesthetized animal (the Einthoven model),which uses ventilation pressure as a surrogate measure of airwayresistance. See, for example, Einthoven (1892) Pfugers Arch. 51:367-445;and Mohammed et al. (2000) Pulm Pharmacol Ther. 13(6):287-92, as well asAssay 3 which describes a rat Einthoven model. Another useful in vivoassay is the rat antisialagogue assay (for example, as described inAssay 4).

Pharmaceutical Compositions and Formulations

A crystalline freebase of the invention is typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. However, it will be understood by those skilledin the art that, once a crystalline freebase of the invention has beenformulated, it may no longer be in crystalline form, i.e., thecrystalline freebase may be dissolved in a suitable carrier.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient and a crystalline freebase of biphenyl-2-ylcarbamicacid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester. The pharmaceutical composition may contain other therapeuticand/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a crystalline freebase ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester, as the active agent. Typically, such pharmaceutical compositionswill contain from about 0.01 to about 95% by weight of the active agent;including, from about 0.01 to about 30% by weight; such as from about0.01 to about 10% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combination of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials that can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols such aspropylene glycol; polyols such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; compressedpropellant gases such as chlorofluorocarbons and hydrofluorocarbons; andother non-toxic compatible substances employed in pharmaceuticalcompositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending the crystalline freebasewith a pharmaceutically acceptable carrier and one or more optionalingredients. If necessary or desired, the resulting uniformly blendedmixture can then be shaped or loaded into tablets, capsules, pills,canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of the invention, a pharmaceutical compositioncomprising the active agent is administered by inhalation using anebulizer inhaler. Such nebulizer devices typically produce a stream ofhigh velocity air that causes the pharmaceutical composition comprisingthe active agent to spray as a mist that is carried into the patient'srespiratory tract. Accordingly, when formulated for use in a nebulizerinhaler, the crystalline freebase active agent is typically dissolved ina suitable carrier to form a solution. Suitable nebulizer devicesinclude the Respimat® Soft Mist™ Inhaler (Boehringer Ingelheim), theAERx® Pulmonary Delivery System (Aradigm Corp.), and the PARI LC PlusReusable Nebulizer (Pari GmbH).

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a crystalline freebase of the invention.In one embodiment, the aqueous nebulizer formulation is isotonic. In oneembodiment, such a solution has a pH of about 4-6. In a particularembodiment, the aqueous nebulizer formulation is buffered with citratebuffer to a pH of about 5. In another particular embodiment, the aqueousformulation contains from about 0.1 mg/mL to about 1.0 mg/mL free baseequivalents of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester.

In another specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a DPI. Such DPIs typically administer the active agent as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the crystallinefreebase active agent is typically formulated with a suitable excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Micronization is acommon method of reducing crystal size to that suitable for pulmonarydelivery. Typically, a crystalline freebase active agent is micronizedand combined with a suitable carrier to form a suspension of micronizedparticles of respirable size, where “micronized particles” or“micronized form” means at least about 90% of the particles have adiameter of less than about 10 m. Other methods of reducing particlesize may also be used such as fine milling, chopping, crushing,grinding, milling, screening, trituration, pulverization, and so forth,as long as the desired particle size can be obtained.

A representative pharmaceutical composition for use in a DPI comprisesdry lactose having a particle size between about 1 m and about 100 m andmicronized particles of a crystalline freebase of the invention. Such adry powder formulation can be made, for example, by combining thelactose with the crystalline freebase active agent and then dry blendingthe components. Alternatively, if desired, the crystalline freebaseactive agent can be formulated without an excipient. The pharmaceuticalcomposition is then typically loaded into a dry powder dispenser, orinto inhalation cartridges or capsules for use with a dry powderdelivery device.

Examples of DPI delivery devices include Diskhaler (GlaxoSmithKline,Research Triangle Park, N.C.; see, e.g., U.S. Pat. No. 5,035,237 toNewell et al.); Diskus (GlaxoSmithKline; see, e.g., U.S. Pat. No.6,378,519 to Davies et al.); Turbuhaler (AstraZeneca, Wilmington, Del.;see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin); Rotahaler(GlaxoSmithKline; see, e.g., U.S. Pat. No. 4,353,365 to Hallworth etal.) and Handihaler (Boehringer Ingelheim). Further examples of suitableDPI devices are described in U.S. Pat. No. 5,415,162 to Casper et al.,U.S. Pat. No. 5,239,993 to Evans, and U.S. Pat. No. 5,715,810 toArmstrong et al., and references cited therein. The disclosures of theaforementioned patents are incorporated herein by reference in theirentirety.

In yet another specific embodiment of the invention, a pharmaceuticalcomposition comprising a crystalline freebase active agent isadministered by inhalation using an MDI, which typically discharges ameasured amount of the active agent using compressed propellant gas.Accordingly, pharmaceutical compositions administered using an MDItypically comprise a solution or suspension of the crystalline freebaseactive agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including chlorofluorocarbons such as CCl₃F,and hydrofluoroalkanes (HFAs) such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due toconcerns about chlorofluorocarbons affecting the ozone layer,formulations containing HFAs are generally preferred. Additionaloptional components of HFA formulations include co-solvents such asethanol or pentane, and surfactants such as sorbitan trioleate, oleicacid, lecithin, and glycerin. See, for example, U.S. Pat. No. 5,225,183to Purewal et al., EP 0717987 A2 (Minnesota Mining and ManufacturingCompany), and WO 92/22286 (Minnesota Mining and Manufacturing Company,the disclosures of which are incorporated herein by reference in theirentirety.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01 to 5% by weight of a freebasecrystalline compound of the invention; from about 0 to 20% by weightethanol; and from about 0 to 5% by weight surfactant; with the remainderbeing an HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing thecrystalline freebase active agent, ethanol (if present) and thesurfactant (if present). To prepare a suspension, the crystallinefreebase active agent is micronized and then combined with thepropellant. The formulation is then loaded into an aerosol canister,which forms a portion of a metered-dose inhaler device. Examples ofmetered-dose inhaler devices developed specifically for use with HFApropellants are described in U.S. Pat. No. 6,006,745 to Marecki and U.S.Pat. No. 6,143,277 to Ashurst et al. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. See, for example, WO 99/53901(Glaxo Group Ltd.) and WO 00/61108 (Glaxo Group Ltd.). The disclosuresof the aforementioned patents and publications are incorporated hereinby reference in their entirety.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. No. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast;U.S. Pat. No. 5,874,063 to Briggner et al.; and U.S. Pat. No. 6,221,398to Jakupovic et al.; and WO 99/55319 (Glaxo Group Ltd.) and WO 00/30614(AstraZeneca AB); the disclosures of which are incorporated herein byreference in their entirety.

In another embodiment, the pharmaceutical compositions of the inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a crystallinefreebase of the invention as an active ingredient. The pharmaceuticalcomposition may be packaged in a unit dosage form.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), a pharmaceutical composition ofthe invention will typically comprise a crystalline freebase of theinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; binders such as carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants such as glycerol; disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and/or sodium carbonate; solution retarding agents such asparaffin; absorption accelerators such as quaternary ammonium compounds;wetting agents such as cetyl alcohol and/or glycerol monostearate;absorbents such as kaolin and/or bentonite clay; lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and/or mixtures thereof; coloring agents; andbuffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically acceptable antioxidants include: water-solubleantioxidants such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; oil-solubleantioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like. Coating agents for tablets,capsules, pills and like, include those used for enteric coatings suchas cellulose acetate phthalate (CAP), polyvinyl acetate phthalate(PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the invention may also beformulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the invention mayoptionally contain opacifying agents and may be formulated so that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The crystalline freebase active ingredient canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent such as,for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (especially cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

A crystalline freebase of the invention can also be administeredtransdermally using known transdermal delivery systems and excipients.For example, the crystalline freebase can be admixed with permeationenhancers such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

A crystalline freebase of the invention can also be co-administered withother therapeutic agents. This combination therapy involves using thecrystalline freebase combined with one or more of these secondaryagents, either formulated together (e.g., packaged together in a singleformulation) or formulated separately (e.g., packaged as separate unitdosage forms). Methods of formulating multiple agents together in thesame formulation or in separate unit dosage forms, are well known in theart. The term “unit dosage form” refers to a physically discrete unitsuitable for dosing a patient, i.e., each unit containing apredetermined quantity of a compound of the invention calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

The additional therapeutic agent(s) can be selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids; non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators).

One particular embodiment of the invention relates to a compositioncomprising (a) a pharmaceutically acceptable carrier and atherapeutically effective amount of a crystalline freebase of theinvention; and (b) a pharmaceutically acceptable carrier and atherapeutically effective amount of an agent selected from a steroidalanti-inflammatory agent such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof; wherein the crystalline freebase and the agent are formulatedtogether or separately. In another embodiment, (b) is a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a β₂adrenergic receptor agonist and a steroidal anti-inflammatory agent. Thesecondary agents can be used in the form of pharmaceutically acceptablesalts or solvates, and if appropriate, as optically pure stereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with a crystalline freebase of the invention include, butare not limited to, salmeterol, salbutamol, formoterol, salmefamol,fenoterol, terbutaline, albuterol, isoetharine, metaproterenol,bitolterol, pirbuterol, levalbuterol and the like, or pharmaceuticallyacceptable salts thereof. Other β₂ adrenergic receptor agonists that canbe used include, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds described in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds described in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds described in WO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds described in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds described in U.S. Pat. No. 6,576,793 to Moran etal.; N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds described in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.When employed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to 500 μg per dose. Thedisclosures of the aforementioned patents and publications areincorporated herein by reference in their entirety.

Representative steroidal anti-inflammatory agents that can be used incombination with a crystalline freebase of the invention include, butare not limited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate, 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3 S-yl) ester, beclomethasone esters(e.g., the 17-propionate ester or the 17,21-dipropionate ester),budesonide, flunisolide, mometasone esters (e.g., the furoate ester),triamcinolone acetonide, rofleponide, ciclesonide, butixocortpropionate, RPR-106541, ST-126 and the like, orpharmaceutically-acceptable salts thereof. When employed, the steroidalanti-inflammatory agent will be present in the composition in atherapeutically effective amount. Typically, the steroidalanti-inflammatory agent will be present in an amount sufficient toprovide from about 0.05 μg to 500 μg per dose.

An exemplary combination is a crystalline freebase of the invention,co-administered with salmeterol as the β₂ adrenergic receptor agonist,and fluticasone propionate as the steroidal anti-inflammatory agent.Another exemplary combination is a e crystalline freebase of theinvention, co-administered with a crystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent. Asnoted above, these agents can be formulated together or separately.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (e.g., sodium cromoglycate,nedocromil sodium, and phosphodiesterase (PDE) inhibitors such astheophylline, PDE4 inhibitors and mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4inhibitors that can be used in combination with a crystalline freebaseof the invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds described inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds described in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with a crystalline freebase of theinvention include, but are not limited to, atropine, atropine sulfate,atropine oxide, methylatropine nitrate, homatropine hydrobromide,hyoscyamine (d, l) hydrobromide, scopolamine hydrobromide, ipratropiumbromide, oxitropium bromide, tiotropium bromide, methantheline,propantheline bromide, anisotropine methyl bromide, clidinium bromide,copyrrolate (Robinul), isopropamide iodide, mepenzolate bromide,tridihexethyl chloride (Pathilone), hexocyclium methylsulfate,cyclopentolate hydrochloride, tropicamide, trihexyphenidylhydrochloride, pirenzepine, telenzepine, AF-DX 116 and methoctramine andthe like, or a pharmaceutically acceptable salt thereof; or, for thosecompounds listed as a salt, alternate pharmaceutically acceptable saltthereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with a crystalline freebase of the inventioninclude, but are not limited to, ethanolamines such as carbinoxaminemaleate, clemastine fumarate, diphenylhydramine hydrochloride anddimenhydrinate; ethylenediamines such as pyrilamine amleate,tripelennamine hydrochloride and tripelennamine citrate; alkylaminessuch as chlorpheniramine and acrivastine; piperazines such ashydroxyzine hydrochloride, hydroxyzine pamoate, cyclizine hydrochloride,cyclizine lactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Unless otherwise indicated, exemplary suitable doses for the othertherapeutic agents administered in combination with a crystallinefreebase of the invention are in the range of about 0.05 μg/day to 100mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the invention, as well as exemplary methods ofpreparation. One or more secondary agents can optionally be formulatedwith a crystalline freebase of the invention (primary active agent).Alternately, the secondary agents(s) can be formulated separately andco-administered with the primary active agent, either simultaneously orsequentially. For example, in one embodiment, a single dry powderformulation can be manufactured to include both the crystalline freebaseof the invention and one or more secondary agents. In anotherembodiment, one formulation is manufactured to contain the crystallinefreebase of the invention and separate formulation(s) are manufacturedto contain the secondary agent(s). Such dry powder formulations can thenbe packaged in separate blister packs and administered with a single DPIdevice.

Exemplary Dry Powder Formulation for Administration by Inhalation

0.2 mg of a crystalline freebase of the invention is micronized and thenblended with 25 mg of lactose. The blended mixture is then loaded into agelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

Exemplary Dry Powder Formulation for Administration by a Dry PowderInhaler

A dry powder is prepared having a bulk formulation ratio of micronizedcrystalline freebase of the invention (active agent) to lactose of1:200. The powder is packed into a dry powder inhalation device capableof delivering between about 10 μg and 100 μg of active agent per dose.

Exemplary Formulations for Administration by a Metered Dose Inhaler

A suspension containing 5 wt % of a crystalline freebase of theinvention (active agent) and 0.1 wt % lecithin is prepared by dispersing10 g of the crystalline freebase as micronized particles with a meansize less than 10 μm in a solution formed from 0.2 g of lecithindissolved in 200 mL of demineralized water. The suspension is spraydried and the resulting material is micronized to particles having amean diameter less than 1.5 μm. The particles are loaded into cartridgeswith pressurized 1,1,1,2-tetrafluoroethane.

Alternately, a suspension containing 5 wt % of a crystalline freebase ofthe invention, 0.5 wt % lecithin, and 0.5 wt % trehalose is prepared bydispersing 5 g of the crystalline freebase as micronized particles witha mean size less than 10 μm in a colloidal solution formed from 0.5 g oftrehalose and 0.5 g of lecithin dissolved in 100 mL of demineralizedwater. The suspension is spray dried and the resulting material ismicronized to particles having a mean diameter less than 1.5 m. Theparticles are loaded into canisters with pressurized1,1,1,2-tetrafluoroethane.

Exemplary Aqueous Aerosol Formulation for Administration by Nebulizer

A pharmaceutical composition is prepared by dissolving 0.5 mg of acrystalline freebase of the invention (active agent) in 1 mL of a 0.9%sodium chloride solution acidified with citric acid. The mixture isstirred and sonicated until the active agent is dissolved. The pH of thesolution is adjusted to a value in the range of from 3 to 8 (typicallyabout 5) by the slow addition of NaOH.

Exemplary Hard Gelatin Capsule Formulation for Oral Administration

The following ingredients are thoroughly blended and then loaded into ahard gelatin capsule: 250 mg of a crystalline freebase of the invention,200 mg of lactose (spray-dried), and 10 mg of magnesium stearate, for atotal of 460 mg of composition per capsule.

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active ingredient per 10 mL of suspension.

Ingredients Amount a crystalline freebase of the invention 1.0 g fumaricacid 0.5 g sodium chloride 2.0 g methyl paraben 0.15 g propyl paraben0.05 g granulated sugar 25.5 g sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g flavoring 0.035 mL colorings 0.5 mg distilledwater q.s. to 100 mL

Exemplary Injectable Formulation

The following ingredients are blended and the pH is adjusted to 4±0.5using 0.5 N HCl or 0.5 N NaOH.

Ingredients Amount a crystalline freebase of the invention 0.2 g sodiumacetate buffer solution (0.4M) 2.0 mL HCl (0.5N) or NaOH (0.5N) q.s. topH 4 water (distilled, sterile) q.s. to 20 mL

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated. The following abbreviations have thefollowing meanings unless otherwise indicated and any otherabbreviations used herein and not defined have their standard meaning:

-   -   AC adenylyl cyclase    -   BSA bovine serum albumin    -   cAMP 3′-5′ cyclic adenosine monophosphate    -   CHO Chinese hamster ovary    -   cM₅ cloned chimpanzee M5 receptor    -   DCM dichloromethane    -   dPBS Dulbecco's phosphate buffered saline    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   FBS fetal bovine serum    -   FLIPR fluorometric imaging plate reader    -   HBSS Hank's buffered salt solution    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hM₁ cloned human M1 receptor    -   hM₂ cloned human M2 receptor    -   hM₃ cloned human M3 receptor    -   hM₄ cloned human M4 receptor    -   hM₅ cloned human M5 receptor    -   HOBT N-hydroxybenzotriazole    -   HPLC high-performance liquid chromatography    -   MCh methylcholine    -   MeCN acetonitrile

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, reagents, startingmaterials and solvents were purchased from commercial suppliers (such asSigma-Aldrich, Fluka, and the like) and were used without furtherpurification.

Preparation 1 Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

The diphosphate salt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (16 g) was dissolved in a biphasic mixture of water (100 mL) andEtOAc (200 mL). NaOH (2 N, 75 mL) was added over a period of 5 minutes.The mixture was then stirred for 30 minutes. The phases were separatedand the aqueous phase was extracted with EtOAc (200 mL). The combinedorganic phases were concentrated. DCM (100 mL) was added, and themixture evaporated to dryness. The solids were dried in an oven forabout 48 hours to yield the title compound (9.6 g).

Example 1 Crystalline Freebase of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form III)

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (102.4 mg) was dissolved in MeCN (500 μL). The solution wasstirred at room temperature for 80 minutes and a white solid precipitateformed. The mixture was placed in the shaker block to thermocycle (0-40°C. in one hour blocks) for 48 hours. A white, dense, immobile solid wasobserved. MeCN (500 μL) was added to mobilize the slurry. The mixturewas then placed back in the shaker block for 2 hours. The solids wereisolated by vacuum filtration using a sinter funnel, then placed in thepiston dryer at 40° C. under full vacuum for 15.5 hours, to yield 76.85mg of the title crystalline compound.

Example 2 Crystalline Freebase of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form III)

Diphosphate salt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoyl-piperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (C₃₅H₄₃N₅O₄.2H₃PO₄; MW 793.75; 632.9 g) was slurried in isopropylacetate (11.08 L) and water (6.33 L) at room temperature under nitrogen.The suspension was warmed to 53±3° C. and 10M NaOH (317 mL) was added tothe stirred mixture, while maintaining the temperature of the mixtureabove 50° C. The mixture was stirred for approximately 5 minutes at53±3° C. before allowing the layers to settle. The layers were thenseparated and the aqueous layer was removed. Water (3.16 L) was added tothe organic layer while maintaining the temperature of the mixture above50° C. The mixture was stirred for 5 minutes at 53±3° C. before allowingthe layers to settle. The layers were separated and the water layer wasremoved. Isopropyl acetate (6.33 L) was added and then about 10 volumesof distillate were collected by atmospheric distillation. This step wasrepeated with additional isopropyl acetate (3.2 L). After the seconddistillation, the temperature of the clear solution was reduced to 53±3°C., then seeded with a suspension of the biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester crystalline freebase (Form III; 3.2 g) in isopropyl acetate (51mL). The resulting suspension was stirred at 53±3° C. for 2 hours, thencooled to 10±3° C. over 4 hours. The suspension was stirred at 10±3° C.for at least 2 hours and then the solids were collected by filtration.The resulting filter cake was washed with isopropyl acetate (2×1.9 L)and the product was dried in vacuo at 50° C. to yield the titlecrystalline compound (C₃₅H₄₃N₅O₄; MW 597.76; 382.5 g, 80.3% yield).

Example 3 Recrystallization of Crystalline Freebase ofBiphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form III)

Crystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (Form III; C₃₅H₄₃N₅O₄; MW 597.76; 372.5 g) was slurried in toluene(5.6 L) at 20±3° C. under nitrogen. The suspension was warmed to 82±3°C., and held at this temperature until complete dissolution wasobserved. The solution was then clarified into the crystallizer vessel,followed by rinsing with toluene (373 μL). Solids were observed in thecrystallizer vessel, and the vessel was re-heated to 82±3° C. to effectdissolution, then cooled to 58±3° C. and seeded with a pre-sonicated(approximately 1 minute) of crystalline freebase (Form III; 1.9 g) intoluene (8 μL). The resulting suspension was allowed to stand at 58±3°C. for at least 4 hours, then cooled to 20±3° C. over 2 hours(approximate cooling rate of 0.33° C./min). The suspension was stirredat 20±3° C. for at least 1 hour, then the solids were collected byfiltration. The resulting filter cake was washed with toluene (2×1.2 L)and the product was dried in vacuo at 52±3° C. to yield the titlecrystalline compound (345.3 g, 92.7% yield).

Example 4 Crystalline Freebase of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form IV)

Biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (prepared as described in Preparation 1; 2.5 g) was dissolved inMeCN (10 mL) to yield a viscous oily pale yellow material. AdditionalMeCN (5 mL) was added to dilute the material. The solution was seededwith crystalline freebase (20 mg; Form III prepared as described inExample 1) and stirred at room temperature for 90 minutes. A largeamount of white precipitate (small crystals) was observed. The slurrywas analyzed under a polarized light microscope and found to bebirefringent.

Additional MeCN (3 mL) was added and the slurry was placed in a MetzSyn10 block to thermocycle (0-40° C. in one hour blocks) at 800 rpmovernight. The Metz Syn10 is a 10 position parallel reaction stationthat is static. Agitation of the solution/slurry was by a cross magneticstirrer bar. The shaker block was a separate piece of equipment that washeated and cooled by an external Julabo bath. The material was removedat 0° C. It was observed that the slurry had settled out, leaving a paleyellow solution above the white precipitate. The slurry was stirred andplaced back in the shaker block to thermocycle. The material was removedat 40° C., and stirred at a high agitation rate at room temperature for80 minutes. The slurry was again analyzed and found to be birefringent.The filter cake was isolated by vacuum filtration using a sinter funnel.MeCN (3 mL) was used to wet the filter paper and the filter cake waswashed with MeCN prior to filtration. The cake was deliquored undervacuum for 40 minutes to yield 2.3 g of a flowing white powder. Thematerial was placed in a piston dryer at 40° C. for 65 hours, to yield2.2 g of the title crystalline compound as a white powder (99.6%purity).

The majority of the Raman spectra of the product was consistent withthat of the Form III starting material. However, three shifts werenoted:

Form III Product 878 cm⁻¹ 881 cm⁻¹ 775 cm⁻¹ 772 cm⁻¹ 485 cm⁻¹ 488 cm⁻¹The product was then analyzed by powder X-ray diffraction, differentialscanning calorimetry, and thermal gravimetric analysis. It wasdetermined that the product was a different freebase crystalline fromthan the Form III starting material, and was designated Form IV.

Example 5 Powder X-Ray Diffraction

Powder X-ray diffraction (PXRD) patterns of the crystalline freebaseForms III (from Example 1) and IV (from Example 4) were acquired on aPANalytical X'Pert Pro powder diffractomer, equipped with an XCeleratordetector. The acquisition conditions were radiation: Cu Kα; generatortension: 40 kV; generator current: 45 mA; start angle 2.0° 2θ; end angle40.0° 2θ, step size: 0.0167° 2θ. The time per step was 31.750 seconds.The sample was prepared by mounting a few milligrams of sample on aSilicon wafer (zero background) plate, resulting in a thin layer ofpowder.

Characteristic peak positions and calculated d-spacings are summarizedbelow, only reporting those peaks with greater than 14% relativeintensity. These were calculated from the raw data using Highscoresoftware. The experimental error in the peak positions is approximately+0.1° 2θ. Relative peak intensities will vary due to preferredorientation.

Form III Form IV Pos. d-spacing Rel. Int. Pos. d-spacing Rel. Int.[°2Th.] [Å] [%] [°2Th.] [Å] [%] 6.6 13.5 53.8 6.6 13.4 27.1 8.8 10.114.8 10.6 8.4 13.7 10.1 8.8 14.1 13.1 6.8 42.0 11.4 7.8 21.7 15.0 5.958.4 11.6 7.6 14.7 16.0 5.5 15.0 13.1 6.8 29.3 17.3 5.1 41.2 14.8 6.015.2 17.7 5.0 45.6 15.2 5.8 15.8 18.6 4.8 100.0 16.1 5.5 30.1 19.7 4.581.2 16.4 5.4 13.9 20.2 4.4 29.7 16.9 5.2 13.8 20.9 4.2 34.8 17.5 5.125.5 21.4 4.1 74.8 18.2 4.9 38.4 22.6 3.9 34.3 18.6 4.8 23.6 24.6 3.618.1 19.3 4.6 23.1 27.8 3.2 16.1 19.7 4.5 100.0 19.9 4.5 73.5 20.2 4.422.8 20.8 4.3 72.7 21.1 4.2 51.5 21.7 4.1 21.7 22.3 4.0 31.0

A representative PXRD pattern for the crystalline freebase Form III isshown in FIG. 1. A representative PXRD pattern for the crystallinefreebase Form IV is shown in FIG. 2.

Example 6 Thermal Analysis

Differential scanning calorimetry (DSC) thermograms of the crystallinefreebase Forms III (from Example 1) and IV (from Example 4) wereobtained using a TA Instruments calorimeter. The samples were weighedinto an aluminum pan, a pan lid placed on top and lightly crimpedwithout sealing the pan. The experiments were conducted using a heatingrate of 10° C./min.

A representative DSC thermograph for the crystalline freebase Form IIIis shown in FIG. 4. The DSC thermograph demonstrates that Form III ischaracterized by a DSC thermograph which shows an onset of endothermicheat flow at 123.1° C. (enthalpy 67.7 J/g).

A representative DSC thermograph for the crystalline freebase Form IV isshown in FIG. 5. The DSC thermograph demonstrates that Form IV ischaracterized by a DSC thermograph which shows a small endotherm and amain endotherm, i.e., a small first onset of endothermic heat flowoccurring at 65.6° C. (enthalpy 0.8 J/g) and a main second onset ofendothermic heat flow occurring at 118.8° C. (enthalpy 66.8 J/g).

Thermal gravimetric analysis (TGA) data were obtained using a TAInstruments Q500 instrument. The samples were heated in an openaluminium pan at a heating rate of 10° C./min to 200° C.

A representative TGA trace for the crystalline freebase Form III isshown in FIG. 6, and indicates that negligible weight loss was observedprior to sample degradation. A representative TGA trace for thecrystalline freebase Form IV is shown in FIG. 7, and indicates thatapproximately 0.3% weight loss was observed prior to sample melt, whichis consistent with the loss of residual solvent.

Example 7 Gravimetric Vapor Sorption Assessment

Gravimetric vapor sorption (GVS) studies were performed using a SurfaceMeasurements System DVS-1 instrument for generation of full sorptionisotherm using water vapor perfusion at 25° C. A sample size ofapproximately 7 mg was placed into a clean and dry tared sample mesh panand weighed using the internal balance. The target relative humidity(RH) ranges were from 30% to 90%, then 90% to 0% and 0% to 30% with 10%steps. The point of equilibrium was automatically determined using a0.02 dm/dt asymptote setting.

GVS studies on a sample of the crystalline freebase Form III conductedat 25° C. demonstrated that the material had a low propensity to take upmoisture over the range 0% RH to 90% RH. The sample showed a reversiblewater uptake of <2% w/w between 0 and 90% RH at 25° C. This GVS tracedemonstrates that Form III has an acceptable weight gain when exposed toa broad humidity range.

Example 8 Micronization

Samples of the crystalline freebase Form III were micronized usingeither an APTM 4″ micronizer and the particle size determined buy laserlight diffraction.

Amt of crystalline Particle size of material micronized material (μm)input (g) yield (g) X₁₀ X₅₀ X₉₀ 60.11 50.73 1.27 2.69 5.25For reference, the particle size of the input crystalline freebase FormIII was X₁₀=5.58 μm X₅₀=18.2 μm, and X₉₀=49.7 μm. Micronization yieldedparticles in the respirable size range. Micronization resulted in areduction in crystallinity but retained the essential characteristics ofthe pre-micronized material. No changes were observed in the PXRD afterstorage for 3 months at 40° C./20% relative humidity, at 40° C./75%relative humidity (uncapped), and at 50° C./ambient humidity.

The DSC thermograph for the crystalline freebase Form III showed a sharpmelt at 125° C., before and after micronization. There was an additionalsmall thermal event in the micronized material at 87° C., likely due tocrystallization. After storage for 3 months at 40° C./20% RH, 40° C./75%RH naked, and 50° C./ambient humidity, the micronized material showed asharp melt at 125° C. with no evidence of amorphous content.

Example 9 Lactose Compatibility

Two formulations of the crystalline freebase Form III were evaluated asto stability for 3 months at 40° C./20% relative humidity (RH), at 40°C./75% RH (uncapped), and at 50° C./ambient humidity. 0.08 wt/wt % (10μg DPI dose) and 2 wt/wt % (250 μg DPI dose) formulations were preparedas a blend with lactose alone, or with lactose and 1 wt/wt % magnesiumstearate. The stability of all formulations was found to be acceptable.

Example 10 pH Solubility and Stability

The crystalline freebase Form III shows good solubility (greater thanapproximately 2 mg/mL) in media up to pH 7. Solubility in water is 0.66mg/mL with a natural pH of 8.9. Solubility in simulated lung fluid is0.46 mg/mL with no change observed between 4 hour and 24 hour solubilitymeasurements.

The crystalline freebase Form III solutions are stable in pH 4 and pH 6buffers for up to 7 days at 50° C. or exposed to light. The solutionsare stable in water and saline for 7 days at room temperature, protectedfrom light.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 1000 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 40 μM in dilution buffer and then serially diluted 5×with dilution buffer to final concentrations ranging from 400 fM to 4μM. The addition order and volumes to the assay plates were as follows:825 μL assay buffer with 0.1% BSA, 25 μL radioligand, 100 μL dilutedtest compound, and 50 μL membranes. Assay plates were incubated for 6hours at 37° C. Binding reactions were terminated by rapid filtrationover GF/B glass fiber filter plates (Perkin Elmer Inc., Wellesley,Mass.) pre-treated in 0.3% polyethyleneimine (PEI). Filter plates wererinsed three times with wash buffer (10 mM HEPES) to remove unboundradioactivity. Plates were then air dried, and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer Inc., Wellesley, Mass.) wasadded to each well. The plates were then counted in a PerkinElmerTopcount liquid scintillation counter (PerkinElmer Inc., Wellesley,Mass.). Binding data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the one-site competition model. K_(i) values for testcompounds were calculated from observed IC₅₀ values and the K_(D) valueof the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff W.H. Biochemical Pharmacology 22(23):3099-108 (1973)). K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forthe M₃ muscarinic receptor subtype when tested in this or a similarassay.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays are performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in Assay 1. The detached cellsare washed twice by centrifugation at 650×g for five minutes in 50 mLsdPBS. The cell pellet is then re-suspended in 10 mL dPBS, and the cellsare counted with a Coulter Z1 Dual Particle Counter (Beckman Coulter,Fullerton, Calif.). The cells are centrifuged again at 650×g for fiveminutes and re-suspended in stimulation buffer to an assay concentrationof 1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 μM indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25μM final concentration diluted in dPBS), 25 μL diluted oxotremorine, and50 μL cells are added to agonist assay wells. To measure the ability ofa test compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS) 25 μL diluted test compound, and 50 μLcells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data are analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively. The K_(i) values are converted topK_(i) values to determine the geometric mean and 95% confidenceintervals. These summary statistics are then converted back to K_(i)values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of oxotremorine-inhibition of forskolin-mediated cAMPaccumulation in CHO-K1 cells expressing the hM₂ receptor, when tested inthis or a similar assay.

Blockade of Agonist-Mediated [³⁵S]GTPγS-Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine isdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates are then incubated at 37° C. for 60 minutes. The assayplates are filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates are rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) is added to each well, and each plate issealed and radioactivity counted on a topcounter (PerkinElmer). Data areanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation is used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of oxotremorine-stimulated [³⁵S]GTPγS-binding in CHO-K1 cellsexpressing the hM₂ receptor, when tested in this or a similar assay.

Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 μL/well of FLIPRbuffer. The cells are then incubated with 50 μL/well of 4 μM FLUO-4AM (a2× solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F){circumflex over ( )}1/H)*EC₅₀. An oxotremorineconcentration of 3×EC_(F) is prepared in stimulation plates such that anEC₉₀ concentration of oxotremorine is added to each well in theantagonist inhibition assay plates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measures thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of agonist-mediated calcium release in CHO cells stablyexpressing the hM₃ receptor, when tested in this or a similar assay.

Assay 3 Rat Einthoven Assay

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity. Alltest compounds are diluted in sterile water and are dosed via theinhalation route (IH). The rats (Sprague-Dawley, male, 250-350 g) areexposed to the aerosol generated from an LC Star Nebulizer Set anddriven by a mixture of gases (5% CO₂/95% atmospheric air). Each testcompound solution is nebulized over a 10 minute time period in a pieshaped dosing chamber capable of holding six rats. At predetermined timepoints after inhalation of compound, the Einthoven assay is performed.

Thirty minutes prior to the start of pulmonary evaluation, the animalsare anesthetized with inactin (thiobutabarbital, 120 mg/kg IP). Thejugular vein is catheterized with saline filled polyethylene catheters(PE-50) and used to infuse MCh. The trachea is then dissected andcannulated with a 14 G needle and used for rat ventilation duringpulmonary evaluation. Once surgery is complete, rats are ventilatedusing a piston respirator set at a stroke volume of 1 ml/100 g bodyweight but not exceeding 2.5 ml volume, and at a rate of 90 strokes perminute.

The changes in pressure that occur with each breath are measured.Baseline values are collected for at least 2.5 minutes then rats arechallenged non-cumulatively with 2-fold incremental increases of thebronchoconstrictor MCh (5, 10, 20, 40 and 80 μg/ml). The MCh is infusedfor 2.5 minutes from a syringe pump at a rate of 2 mL/kg/min. Theanimals are euthanized upon completion of the studies.

Changes in ventilation pressure (cm H₂O) in treated animals areexpressed as % inhibition of MCh response relative to control animals.In this assay, a higher % inhibition value indicates that the testcompound has a bronchoprotective effect. The compound of formula I, whentested in this assay at a dose of 100 μg/ml, is expected to exhibitgreater than 35% inhibition, possibly greater than 70% inhibition, andeven more possibly greater than 90% inhibition.

1.5 hr ID₅₀ Determination

Standard muscarinic antagonists were evaluated in the rat Einthovenassay 1.5 hrs post-dose. The order of potency (ID₅₀s) for the fivestandards tested was determined to be: ipratropium (4.4μg/ml)>tiotropium (6 μg/ml)>des-methyl-tiotropium (12μg/ml)>glycopyrrolate (15 μg/ml)>LAS-34237 (24 μg/ml). The potency ofthe test compound is similarly determined at 1.5 hrs post-dose.

6 and 24 hr ID₅₀ Determination

Standards tiotropium and ipratropium were also evaluated 24 hr and/or 6hr post-dose in the rat Einthoven assay. Ipratropium (10 and 30 μg/ml)was about 3-fold less potent 6-hr post-dose compared to its 1.5 hrpotency. The observed loss of activity at this time point (6 hr) isconsistent with its relatively short duration of action in the clinic.Tiotropium showed a slow onset of effect with peak bronchoprotectionbeing achieved 6-hr post-dose. Its 6 hr and 24 hr potency values werenot significantly different from each other and were about 2-fold morepotent compared to its 1.5 hr potency. The onset of action of the testcompound, as well as the 6 and 24 hr potency values, is similarlydetermined.

Assay 4 Rat Antisialagogue Assay

Rats (Sprague-Dawley, male, 250-350 g) are dosed, anesthetized andcannulated as described for Assay 3. At predetermined time points andafter surgery, animals are placed on their dorsal side at a 200 inclinewith their head in a downward slope. A pre-weighed gauze pad is insertedin the animal's mouth and the muscarinic agonist pilocarpine (PILO) (3mg/kg, iv.) is administered. Saliva produced during 10 minutes post-PILOis measured gravimetrically by determining the weight of the gauze padbefore and after PILO. Antisialagogue effects are expressed as %inhibition of salivation relative to control animals.

1, 6 and 24 hr ID₅₀ Determination

The rat antisialagogue assay was developed to assess systemic exposureand calculate the lung selectivity index (LSI) of test compounds. Thestandard, tiotropium, was evaluated in this model at 1, 6, and 24 hrpost-dose. Tiotropium was found to be most potent at inhibitingpilocarpine-induced salivation 6 hrs post dose. This finding isconsistent with the peak effects observed in the Einthoven assay.

This model is a modified version of the procedure described in Rechter,“Estimation of anticholinergic drug effects in mice by antagonismagainst pilocarpine-induced salivation” Ata Pharmacol Toxicol 24:243-254(1996). The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose.

Exemplary compounds of the invention that are tested in this assay areexpected to exhibit ID₅₀ values less than 100 μg/ml (measured at 24hours), with some compounds expected to exhibit an ID₅₀ value less than30 μg/ml, some less than 20 μg/ml, and some less than 15 μg/ml.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A crystalline freebase of biphenyl-2-ylcarbamicacid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester having a melting point of about 125° C.
 2. A crystalline freebaseof biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester having a differential scanning calorimetry thermogram inaccordance with that shown in FIG.
 4. 3. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and crystallinefreebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester having a melting point of about 125° C.
 4. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier andcrystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester having a differential scanning calorimetry thermogram inaccordance with that shown in FIG. 4.